修改意见 - 第三军医大学学报.doc

新生大鼠原代心房肌细胞钙超载模型的建立 丁 翔，仝识非，秦 瑶，宋治远▲（400038 重庆，第三军医大学西南医院心内科） [摘要] 目的 探讨建立新生大鼠原代心房肌细胞钙超载模型的方法。方法 消化法分离新生大鼠心房肌细胞并行原代培养。不同浓度ionomycin（00.5、1.0、2.0 μmol/L），以钙离子指示剂Fluo-3/AM负载心房肌细胞，激光扫描共聚焦显微镜检测心房肌细胞游离钙离子浓度[Ca2+]i），建立新生大鼠心房肌细胞不同程度钙超载模型。结果 ①培养至第4天，细胞生长密度可以达到瓶底70%左右免疫化学鉴定90%以上培养细胞α-肌动蛋白抗体染色阳性。②对照组心房肌细胞活性比率为73.00士2.37%，各间心房肌细胞活性无显著性差异（0.05）。③经不同浓度ionomycin处理l h后，实验心房肌细胞[Ca2+]i明显增高，与对照组有显著性差异（±20.97、705.87±28.34、1305.05±53.69 vs 257.08±18.63, P0.05），且各实验组间心房肌[Ca2+]i水平随ionomycin浓度的增加而上升（0.05）。光镜下形态学观察表明，随ionomycin浓度的增加，荧光强度，呈浓度依赖性。结论ionomycin能够建立可靠的心房肌细胞钙超载模型。 The study of calcium overload model with primary atrial myocytes of neonate rats Ding Xiang, Tong Shifei, Qin Yao, Song Zhiyuan （Department of Cardiology, Southwest Hospital, Chongqing, 400038, China） [Abstract] Objective To investigate the modeling of calcium overload with primary atrial myocytes of neonate rats. Methods The atrial myocytes were disassociated from neonate rats with digestion method and cultured for 96 hours. Co-incubated with the ionomycin（0、0.5、1.0、2.0 μmol/L, respectively）, the cells were divided into control group and experiment group（E1, E2 and E3） and loaded with Ca2+ indicator Fluo-3 /AM. The level of intracellular Ca2+ ([Ca2+]i) were measured with laser confocal scanning microscope. Results ① More than 90% of cultured cells were positive to α-actin antibody. ② The activity ratio of cells in control group was 73.00士2.37%. Compared with the each subgroup of experiment, there was no significant difference(p0.05). ③ One hour after treated with different concentrations of ionomycin, the [Ca2+]i of atrial muscle cell was significantly increased in all the experimental groups（431.54±20.97, 705.87±28.34, 1305.05±53.69 vs 257.08±18.63, respectively, p0.05）. There was significant difference between each subset of the experimental group. [Ca2+]i level and the fluorescence intensity of the myocytes increased gradually in a dose-dependent manner with the concentration of ionomycin. Conclusion calcium